L0301P37 - Recombinant DNA Techniques and Diagnostic DNA tests
Recombinant DNA Technology Recombinant DNA *DNA made of different sequences from different sources Cleaving and Rejoining DNA *requires knowledge of DNA transcription, translation and replication Restriction Enzymes *naturally made by microbes as a defence mechanism against viruses and other foreign DNA **its own DNA is protected by methylation *binds to DNA at specific sequences and cuts it (act as “molecular scissors”) *make two kind of cuts: **staggered cuts - sticky ends **blunt ends *cuts the phosphodiester in the backbone of DNA, H bonds are still present (denatured at high temperatures) *when fragments cut by the same restriction enzyme are mixed, recombinant DNA is formed by complementary base pairing **however DNA ligase is required to reform the phosphodiester bonds Analysis of Fragments Gel Electrophoresis *DNA fragments generated from cleavage by restriction enzymes can be separated by size by gel electrophoresis *DNA is negatively charged and migrate towards the positive pole *longer fragments migrate more slowly as they get “tangled” in the agarose gel *slices of the gel can be cut out and the DNA fragment purified for use Hybridisation and Probe *particular fragments can be identified by hybridisation with a probe which is often labeled with fluorescent markers or radioisotope *process: **transfer DNA onto a nylon filter **denature DNA into single strands by making environment basic **mixed with probe (DNA with a known sequence - often a mutation) which will hybridise and markers can be seen Cloning Genes Vectors *hosts for recombinant DNA experiments: bacteria, yeasts and cultured plant cells *newly introduced DNA must be part of a DNA molecules often a plasmid *must contain: **a replication unit (for propagation in host cells) **recognition sequences for restriction enzymes (at which to insert new DNA) ***same restriction enzyme would be used on the recombinant DNA **reporter genes (markers) to identify their presence in host cells, e.g.: ***antibiotic resistance genes ***genes that produce colour under particular conditions ***specialised vectors are used for different hosts Ways to Introduce Foreign DNA *DNA usually cannot cross cell membrane *methods of inserting DNA into a host cell: **virus (used often for plant cells) **chemical treatment - high Ca+ (more porous bacterial membrane) **liposomes - lipid coating over DNA **electroporation - high voltage current pulses create pores in the membrane **microinjection (often used for mammalian cells) **gene gun - bombardment with DNA coated particles (often for plant cells) Selectable Markers *used to identify which cells contain the recombinant DNA from vectors **transformed or transfected (animal) Types: 1. Colour Marker *LacZ gene **based on colour production **codes for enzyme that converts substrate X-Gal into a bright blue product **DNA inserted into LacZ gene - transgenic cells will not be convert X-Gal and will produce white colonies *green fluorescent protein **GFP inserted into a plasmid will emit (green) visible light when exposed to UV light **avoids use of antibiotics 2. Antibiotic Resistance Markers *antibiotic resistance genes encode for proteins that break down antibiotics *only cells that are transformed by the vector that contains the antibiotic resistance gene can grow on medium containing the antibiotic *e.g. ampicillin, tetracycline Sources of Genes for Cloning Genomic Library *a collection of the total genomic DNA from a single organism stored in a population of identical vectors, each containing a different insert of DNA *the cutting of the an organism’s total genome by a restriction enzyme produces many fragments that can be individually and randomly combined with a vector and inserted into a host to create a gene library *a gene (genomic) library represents all genes of an organism Complementary DNA (cDNA) Library *the mRNAs produced in a certain tissue at a certain time can be extracted and used to create cDNA by reverse transcription (enzyme reverse transcriptase) *the cDNAs can then be cloned just like gene fragments to make a cDNA library *it represents only those genes being expressed when the RNA was obtained **i.e. no introns are included Recombinant DNA in Animals Transgenic Mice *heavily used by scientists to study the function of different genes and test different disease models and drugs *are identified by testing genomic DNA for the presence of the transgene by hybridisation testing or PCR *breeding can produce animals homozygous for the transgene *these animals generally have extra genetic information, i.e. “gene addition” Creation Process *created by microinjection of a DNA construct (transgene) into the male pronucleus of fertilised eggs *injected eggs are cultured and transferred to foster mothers where they either: **develop to term **integrate the transgene into their germ line DNA Gene Knockout *homologous recombination (HR) **can be used to selectively inactivate or “knock out” a gene in some organisms, e.g. yeast, mice **occurs during meiosis or as part of the DNA repair process *gene of interest is cloned into a vector, with a reporter gene in the middle of the normal allele (target gene) *recombinant vector is microinjected to transfect mouse embryonic stem cells *sequences line up with homologous pair during meiosis I *if recombination occurs, the normal gene is lost *transfected stem cell with the desired gene modification is transplanted into an early mouse embryo *the mouse and its progeny will have the inactive gene in all cells *can be inbred to produce a homozygous ‘knock-out’ line of mice *phenotypic changes provide clues to the normal allele function Applications of Transgenic and Knockout Animals *transgenic “gene addition” animals are useful: **as human disease models **for testing therapies ***e.g. the Alzheimer mouse expresses the amyloid protein precursor *homozygous knock-out mice: ** also serve as models of human genetic diseases **can be used to assess the roles of genes during development Polymerase Chain Reaction (PCR) *PCR is used to repeatedly replicate DNA in the test tube *single cycle doubles the amount of DNA *with enough primer, DNA polymerase, and substrate dNTPs, repeating the cycle many times leads to a geometric increase in the number of copies of DNA (n cycles = 2n copies of the starting DNA) *PCR requires known sequence at the 3’ end of the DNA to be amplified Process *PCR cycles through three main steps: #heat double-stranded DNA to denature it into single strands #add primers¹ specifically complementary to the 3’ ends of each denatured strand #*mix in dNTPs and DNA polymerase #warm the solution to allow DNA polymerase to elongate the strand and synthesise new DNA ¹ Primers are: *short DNA oligonucleotides (15-20 bases long) *chemically synthesised *designed to achieve high specificity Diagnostic Tests Detecting Human Genetic Variation *specific biochemical treatments and possible cures depend on knowing the molecular basis for human genetic diseases *diagnosis may be possible before symptoms first appear, thus making medical intervention possible Genetic Screening and Tests *detects human gene mutations directly at the DNA level *advantage of testing DNA for mutations directly is that any cell can be tested at any time in the life cycle *however, genetic screening techniques also raise ethical questions about their use Pre-Natal Testing *chorionic villus sampling **8-10th week of pregnancy **DNA extracted from cells of the foetal origin *amniocentesis **13-17th week of pregnancy **foetal cells from the amniotic fluid Heterozygosity Screening *DNA tests are also used to identify people who are predisposed to, or carriers (heterozygotes) of, certain diseases *this knowledge can be used in the best interest of individuals and their offspring. DNA Testing Methods *routine methods: **allele-specific cleavage **allele-specific oligonucleotide hybridisation **allele-specific PCR *these methods all rely on a knowledge of the exact mutation being detected Allele-Specific Cleavage *the base changes responsible for the gene defect may also alter a restriction site *the fragments generated by cutting DNA with the relevant restriction enzyme have different lengths for normal and mutated DNA (gel electrophoresis to check) Allele-Specific Oligonucleotide Hybridisation *oligonucleotides are made that will hybridise to denatured DNA of either the normal or mutated gene *if the probe is radioactively or fluorescently labeled, hybridisation is readily detected *easier and faster than allele-specific cleavage and can detect nay known sequence change Allele-Specific PCR *two oligonucleotide primers are used that differ at the last base; one matches normal, the other matches mutant *only if the primer finds a perfect match in the DNA sample will a PCR product be generated *results: **normal: only normal primer will produce **mutant: only mutant primer will produce **heterozygote: both primers will produce Diagnosing Infectious Disease *tests based on DNA probes and PCR can be used to show whether the DNA of an infectious agent is in blood or a tissue sample *small amount of target sequence (template) is required (e.g. drop of blood) *specificity of primers (must match pathogen DNA) means tests are extremely sensitive Testing for HIV Infection *HIV = Human Immunodeficiency Virus *traditional test detects antibodies to the virus (chance of a false negative result) meaning that symptoms generally have begun to show and the body is having an immune response *during infection, the viral genome is integrated into chromosomes of T-lymphocytes in blood *DNA extracted from a blood sample can be analysed by PCR in combination with a tagged hybridisation probe *advantages: **extremely sensitive **earlier detection and treatment **better prevention of transmission **allows screening of newborns   DNA Fingerprinting *each person’s genome is unique  ∴ individuals can be definitively characterised by their DNA sequence **approximately 2% of the genome is unique between people *routine sequencing of whole genomes (3x109 nucleotides) is becoming increasingly feasible *uses sequences that are highly variable (polymorphic) **have multiple forms in the human population and therefore will be different in each individual Types of Inherited Sequence *single nucleotide polymorphisms (SNPs) **single mutation in each person **very easily distinguishable *short tandem repeats (STR) **repetitive DNA sequences giving rise to side-by-side repeats in the chromosome **number of repeats differ between people **several different STRs are used to generate unique patterns Process *two Processes possible Restriction Enzymes #DNA cut with restriction enzymes that cut at sites that flank the repeat sequences #resultant fragment separated by gel electrophoresis and blotted onto a membrane #pattern is revealed by hybridisation allowing comparison to be made between samples PCR #STR regions amplified by PCR using specific primers #primers hybridise to target sequences flanking the repeat sequences Uses *DNA matching technology *criminal investigations *paternity / Family relatedness *forensic pathology Sources *DNA is extracted from blood, semen, hair, or tissue samples *DNA samples from a crime scene can determine whether a particular suspect left that sample at the scene FBI *uses 13 STR loci in its combined DNA Index System database (CODIS) *with all the alleles and 13 loci, the probability of two people sharing the same alleles is very small